The ethanol-inducible cytochrome P450 2E1 catalyzes the oxidation of a large number of drugs, hepatotoxic xenobiotics, and carcinogens. The primary goals of the proposed research are to gain a better understanding of the active site structure of P450 2E1 and to identify the critical amino acid residues in the active site of 2E1 that are involved in catalysis and substrate binding. High performance liquid chromatography (HPLC) and electrospray ionization liquid chromatography-mass spectrometry (ESI-LC-MS) will be used to determine the identities of the metabolites produced during the mechanism-based inactivation of cytochromes P450 2E1 and 2E1 T303A by tert-butyl acetylene (tBA) and tert-butyl 1-methyl-2-propynyl ether (tBMP). LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry) will be used to identify the tBA-modified polypeptide(s) and amino acid residue(s) in the P450 2E1 active site. NMR methodology and site-specific 2E1 mutants will be used to explore the novel mechanism for the inactivation of P450 2E1 T303A by tBA. Finally, ESI-LC-MS will be used to determine what effect alternate oxidants may have on the mechanism of inactivation of P450s 2E1 and 2E1 T303A by tert-butyl acetylenes. Understanding the active site structure of P450s and identifying the critical amino acid residues involved in catalysis and substrate binding will prove to be extremely valuable for developing techniques that can be used to selectively modulate the catalytic activity of these enzymes.